1. Field of the Invention (Technical Field)
The present invention relates to a novel oryzacystatin protease inhibitor peptide, including a signal peptide component thereof, nucleic acid sequences encoding the protein and signal peptide, incorporation of the oryzacystatin protease inhibitor gene into the genome of a plant and the expression of the inhibitor gene in plants whereby such plants are less susceptible to insect and plant pest damage, and to damage caused by certain plant viruses. The invention is further directed to methods for producing the complete oryzacystatin protease inhibitor peptide, compositions containing the complete oryzacystatin protease inhibitor peptide for use as insecticidal agents, and the use of oryzacystatin protease inhibitor peptide for decreasing insect and virus damage to plants.
2. Background Art
There is significant interest in the use of materials and methods to control insect damage to commercially valuable crop plants that does not require the use of conventional chemical insecticides and chemical fumigation methods. One approach that has been studied employs the use of protease or peptidase inhibitors that are toxic to or substantially inhibit certain insects.
Proteases and peptidases are enzymes that hydrolyze the peptide bonds of proteins or peptides, respectively. These proteases or peptidases (collectively referred to as proteases) play a number of roles in biochemical regulation of organisms, including insects. For example, peptides are generated from proteins by the action of proteases in the gastrointestinal tract of organisms. Through use of these proteases, proteins are broken down into absorbable peptides or individual amino acids, thereby providing sustenance to the organism. Disruption of protease activity can thus have a significant adverse effect on the life cycle of organisms.
There are a number of mechanisms that regulate protease enzymatic activity; one of the most potent and direct mechanisms is through the use of protease inhibitors. Based on the catalytic mechanism employed, there are four classes of protease enzymes, of which one such class is cysteine or thiol proteases. Cysteine proteases are widely distributed, and occur in plants, animals, bacteria and a wide variety of microorganisms. These organisms include specifically plant and animal pests and parasites, as described generally in U.S. Pat. Nos. 5,494,813 and 5,863,775.
Cystatins are well known inhibitors of cysteine proteases. A wide variety of cystatin-type inhibitors have been documented, and these are naturally expressed in a wide variety of plants and animals as part of the regulatory scheme of proteolytic activity. Thus cystatins are naturally found in chickens, including egg whites, U.S. Pat. No. 5,212,297; humans, U.S. Pat. No. 5,919,658; rice and maize, U.S. Pat. No. 5,863,775; and a wide variety of other plants and animals.
Research on the use of cystatins as a means of crop pest control has grown due to the wide spectrum of activity these proteinacious inhibitors possess. The cloning of oryzacystatin-I (OC-I), one of the earliest characterized cystatins of plant origin (Abe et al., Purification of a cysteine proteinase inhibitor from rice, Oryza sativa. Agric Biol Chem 49: 3349-3350 (1985)), was first published over a decade ago (Abe et al., Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). J Biol Chem 262: 16793-16797 (1987); GenBank accession M29259), with other plant cystatins quick to follow, including oryzacystatin-II (OC-II) (Kondo et al., Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. J Biol Chem 265: 15832-15837 (1990)), corn cystatin-I (CC-I), corn cystatin-II (CC-II) (Abe et al., Structural organization of the gene encoding corn cystatin. Biosci Biotech Biochem 60: 11731-1175 (1996)), and a cystatin from sorghum (Li et al., Direct Submission to GenBank, Accession # 1076759, PID g1076759). These cystatins share a high degree of similarity and contain a highly conserved sequence, Gln-Val-Val-Ala-Gly (SEQ ID NO: 7) believed to be the active region of the inhibitor responsible for binding cysteine proteases (Abe et al., The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. J Biol Chem 263: 7655-7659 (1988)).
To date, the transformation of plants with OC-I cDNAs has resulted in low or inconsistent protein yields (Masoud et al., Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants. Plant Mol Biol 21: 655-663 (1993); Irie et al., Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases. Plant Mol Biol 30: 149-157 (1996)). There are reports that plants transformed with OC-I do not significantly hinder the growth of certain crop pests, such as the Coleoptera, Phaedon cochleariae, and may actually cause the pests (Psylliodes chrysocephala L. and Ceutorhynchus assililis) to thrive.
Low protein yields of OC-I occurred even when the cDNA was inserted back into the source plant, Oryza sativa L. japonica, for the purpose of enhancing the effectiveness of the innate OC-I against pests (Irie et al., supra). Thus it has not heretofore been possible to express consistent and high protein yields of OC-I using cDNA transformation methods with the heretofore-identified gene segments.
A variety of different methods have been attempted to employ transformation schemes that result in commercially and agriculturally viable expression of useful levels of OC-I. The earliest report was by Masoud et al. (Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgentic tobacco plants. Plant Mol Biol 21: 655-663 (1993)). Attempts have been made to stabilize OC-I in plants through such approaches as engineering fusion proteins using genes from cowpea trypsin inhibitor (CPTI) (Urwin et al., Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs. Planta 204: 472-479 (1998), beta-glucuronidase (Hosoyama et al., Introduction of a chimeric gene encoding an oryzacystatin-xcex2-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants. Plant Cell Reports 15: 174-177 (1995)), Bacillus thuringiensis Cry 3A (Klypina et al., A chimeric oryzacystatin-I/Bacillus thruingiensis Cry3A gene which is expressed at high levels in transgenic plants, Abst. 330, ASPP p. 84 (1998)) and the 10 kDa zein seed storage protein from maize (Kikuta-Oshima et al., A 10 kDa zein/oryzacystatin-I protease inhibitor chimeric gene designed for the stabilization of proteins for the purpose of plant pest control is expressed in transgenic plants. Abst. 340, ASPP, p. 86 (1998)). Although these strategies seem to improve the stability of OC-I accumulation in transgenic plants, it is unclear whether the resulting fusion proteins retain any cystatin activity.
It has been heretofore universally known that oryzacystatins, unlike the maize cystatins, have no N-terminal leader sequences, and therefore probably remain cystostolic after translation. This assumption was based, in significant part, upon a protein produced by the OC-I gene reported by Abe et al. (1987) and Kondo et al. (Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin. Gene 81: 259-265 (1989)).
Since its discovery, OC-I has been investigated for use as a crop pest deterrent primarily because of its function as an insect digestive system protease inhibitor (see, e.g., U.S. Pat. Nos. 5,863,775 and 5,494,813, Irie et al., supra.). The belief that OC-I is a cytosolic protein was considered important in the work of Urwin et al. (Engineered oryzacystatin-I expressed in transgenic having roots confers resistance to Globodera pallida. Plant J 8: 121-131 (1995)), since nematodes appear to feed only on the cytosol. This belief was supported by the report that OC-I did not appear to have a functional signal peptide (Abe et al. 1988, supra). Several groups have attempted to express the OC-I gene in plants expecting accumulation of OC-I to be sufficient for plant defense, but reproducible levels of accumulation remained elusive. Based on this disclosure of this invention, the instability of prior art OC-I
There is also interest in the use of cysteine protease inhibitors to engineer resistance against viruses, including certain potyviruses. (Gutierrez-Campos et al., The use of cystein proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nature Biotech 17: 1223-1226 (1999)). The processing mechanism of certain viruses, including all known potyviruses, involves the activity of cysteine proteases. Preliminary research has demonstrated that there is a correlation between the level of OC-I message and resistance against viruses with processing mechanisms involving cysteine proteases, such as tobacco etch virus and potato virus Y. Thus effective methods to induce high levels of OC-I expression in plants may afford significant protection against viruses with processing mechanisms involving cysteine proteases, as well as affording protection against plant pests, including insects and nematodes, which utilize cysteine proteases in endogenous metabolic pathways.
Thus there is a significant need for methods and reagents which will result in expression of consistent and high levels of OC-I in various plants, and which result in levels of OC-I expression which are effective for protection against both plant pests and viruses susceptible to cystatin inhibition.
The invention includes an oryzacystatin-I protease inhibitor peptide that includes the amino acid sequence of SEQ ID NO: 1 and sequences that are functionally homologous with the amino acid sequence of SEQ ID NO: 1. It further includes an isolated pre-oryzacystatin-I peptide called SPOC-I that is characterized by the amino acid sequence of SEQ ID NO: 4 and sequences that are functionally homologous with the amino acid sequence of SEQ ID NO: 4. The invention includes a recombinant DNA molecule that includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 4. The invention further includes transgenic plants that include a foreign nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 4 and sequences that are functionally homologous with the amino acid sequence of SEQ ID NO: 4.
The invention also includes a nucleic acid sequence encoding both a signal peptide for OC-I and an OC-I peptide. Transgenic plants that include a foreign nucleic acid sequence encoding both the signal peptide OC-I and the OC-I peptide are included within the invention. The nucleic acid sequence may be a recombinant DNA molecule. The recombinant DNA molecule may be characterized or described in that it is a vector. The invention includes protein, including purified peptide, which is derived from the expression of a nucleic acid sequence encoding both a signal peptide for OC-I and an OC-I peptide. In one embodiment, the nucleic acid sequence includes that set forth in SEQ ID NO: 3 and sequences functionally homologous with the sequence set forth in SEQ ID NO: 3. Host cells may be transformed with vectors that are recombinant DNA molecules encoding both a signal peptide for OC-I and an OC-I peptide. Representative host cells include maize, sweet corn, squash, melon, cucumber, sugarbeet, sunflower, rice, cotton, canola, sweet potato, bean, cowpea, tobacco, soybean and alfalfa cells.
In another embodiment, the invention includes a cystatin protease inhibitor peptide having an amino acid sequence extending from amino acid position 13 to 37 of SEQ ID NO: 1 and at least a portion of the sequence extending from amino acid position 1 to 12, and peptides that are functionally equivalent, such as peptides that have an amino acid sequences functionally homologous to amino acid position 13 to 37 of SEQ ID NO: 1 and that further contain at least a portion of the sequence extending from amino acid position 1 to 12. Also included are recombinant DNA molecules that include a nucleic acid sequence encoding a peptide having an amino acid sequence extending from amino acid position 13 to 37 of SEQ ID NO: 1 and at least a portion of the sequence extending from amino acid position 1 to 12, and peptides that are functionally equivalent. Transgenic plants that include a foreign nucleic acid sequence encoding a peptide having an amino acid sequence extending from amino acid position 13 to 37 of SEQ ID NO: 1 and at least a portion of the sequence extending from amino acid position 1 to 12, and peptides that are functionally equivalent are included within the invention.
The OC-I peptide of this invention may be an homogenous protease inhibitor OC-I peptide characterized by a molecular weight of about 12.6 kDa on an SDS PAGE with binding affinity to polyclonal antibodies to recombinant OC-I.
The invention further provides a method of combating insect pests which includes exposing pests to an insecticidally effective amount of an OC-I peptide, wherein the peptide is expressed in a plant or plant colonizing microorgranism as a result of genetic transformation by a nucleic acid sequence encoding both a signal peptide for OC-I and an OC-I peptide. The plant may be a maize, sweet corn, squash, melon, cucumber, sugarbeet, sunflower, rice, cotton, canola, sweet potato, bean, cowpea, tobacco, soybean or alfalfa plant.
The invention further provides a method of combating viruses with processing mechanisms involving cysteine proteases, which method includes exposing viruses to a viricidally effective amount of an OC-I peptide, wherein the peptide is expressed in a plant as a result of genetic transformation by a nucleic acid sequence encoding both a signal peptide for OC-I and an OC-I peptide. The plant may be a maize, sweet corn, squash, melon, cucumber, sugarbeet, sunflower, rice, cotton, canola, sweet potato, bean, cowpea, tobacco, soybean or alfalfa plant.
A primary object of the present invention is the expression of useful levels of OC-I in crop plants, including, but not limited to, crop plants in which OC-I is naturally expressed, such that the crop plant is effectively resistant to plant pests and viruses susceptible to cystatin inhibition.
Another object of the present invention is to provide the complete genomic DNA for OC-I, including that portion coding a signal peptide, such that transformed eukaryotic organisms express high levels of biologically active OC-I.
Another object of the present invention is to provide an OC-I peptide that is appropriately 12 residues larger than previously described, comprising the peptide sequence (utilizing conventional single letter codes) EAHRAGGEGEEKMSSDGGPVLGGVEPVGNENDLHLVD (SEQ ID NO: 1), which peptide may be expressed in transformed organisms using genomic DNA.
A primary advantage of the present invention is that it provides heretofore unknown genomic DNA encoding for functional OC-I in transformed eukaryotic organisms, including a signal peptide for OC-I necessary for processing the encoded pre-protein into a mature and function form of OC-I.
Another advantage of the present invention is that it provides efficient and effective methods for producing functional OC-I.
Other objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.